Evaluating the role of macrocycles in the susceptibility of hepatitis C virus NS3/4A protease inhibitors to drug resistance

Abstract

The hepatitis C virus (HCV) infects an estimated 150 million people worldwide and is the major cause of viral hepatitis, cirrhosis, and liver cancer. The available antiviral therapies, which include PEGylated interferon, ribavirin, and one of the HCV NS3/4A protease inhibitors telaprevir or boceprevir, are ineffective for some patients and cause severe side effects. More potent NS3/4A protease inhibitors are in clinical development, but the long-term effectiveness of these drugs is challenged by the development of drug resistance. Here, we investigated the role of macrocycles in the susceptibility of NS3/4A protease inhibitors to drug resistance in asunaprevir, danoprevir, vaniprevir, and MK-5172, with similar core structures but varied P2 moieties and macrocyclizations. Linear and macrocyclic analogues of these drugs were designed, synthesized, and tested against wild-type and drug-resistant variants R155K, V36M/R155K, A156T, and D168A in enzymatic and antiviral assays. Macrocyclic inhibitors were generally more potent, but the location of the macrocycle was critical for retaining activity against drug-resistant variants: the P1-P3 macrocyclic inhibitors were less susceptible to drug resistance than the linear and P2-P4 macrocyclic analogues. In addition, the heterocyclic moiety at P2 largely determined the inhibitor resistance profile, susceptibility to drug resistance, and the extent of modulation by the helicase domain. Our findings suggest that to design robust inhibitors that retain potency to drug-resistant NS3/4A protease variants, inhibitors should combine P1-P3 macrocycles with flexible P2 moieties that optimally contact with the invariable catalytic triad of this enzyme.

Figure 1

Structures of NS3/4A protease inhibitors: (A) FDA approved drugs telaprevir and boceprevir; (B) asunaprevir (1) and P1–P3 macrocyclic analogue Asu-mcP1P3 (1a); danoprevir (2), linear analogue Dan-linear (2a), vaniprevir (3), and P1 olefin analogue Dan-mcP2P4 (3a); MK-5172 (4), linear analogue 5172-linear (4a) and P1–P3 macrocyclic analogue 5172-mcP1P3 (4b); the canonical nomenclature for drug moiety positioning is indicated using telaprevir and asunaprevir; arrows indicate corresponding analogues. Asunaprevir, danoprevir/vaniprevir and MK-5172 inhibitor series, which share the same P2 moiety but have different macrocylization status, are indicated by orange, green and blue, respectively.

Figure 2

Synthesis of designed protease inhibitors; reagents and conditions: (a) HATU, DIEA, DMF, CH₂Cl₂; (b) HCl, MeOH; (c) HATU, DIEA, DMF; (d) Zhan 1B catalyst, 1,2-DCE; (e) Potassium vinyltrifluoroborate, TEA, PdCl₂(dppf)-CH₂Cl₂, EtOH; (f) H₂, 10% Pd/C, MeOH, dioxane; (g) 4N HCl, dioxane; (h) Boc-Tle-OH, HATU, DIEA, DMF; (i) LiOH, THF, H₂O; (j) HATU, DMAP, DIEA, DMF, CH₂Cl₂.

Figure 3

(a) Enzyme inhibition constants for HCV genotype 1a full-length NS3/4A and isolated protease domain (dark and light bars) and (b) replicon-based half maximal inhibitory concentrations for genotype 1b HCV NS3/4A and drug resistant variants for (first row) telaprevir and asunaprevir series, (second row) danoprevir/vaniprevir series, and (third row) MK-5172 series. Error bars represent standard errors of the mean (n = 4); * indicates Ki and IC₅₀ values are greater than the highest inhibitor concentration tested.

Figure 4

Resistance profiles of protease inhibitors in (a) enzyme inhibition and (b) cell-based replicon assays for (first row) asunaprevir series, (second row) danoprevir/vaniprevir series, and (third row) MK-5172 series. Enzyme inhibitory and antiviral activities against mutants were normalized with respect to the wild-type full-length HCV NS3/4A protease, NS3/4A protease domain or wild-type HCV clone. Error bars represent propagated standard errors.

Figure 5

Fold-change in inhibitor potencies against full-length NS3/4A compared to the isolated HCV NS3/4A protease domain; values lower than one indicate higher inhibitor potency against the full-length protein compared to protease alone, and vice versa.